Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Structural basis for the design of allosteric inhibitors of the Aurora kinase A enzyme in the cancer chemotherapy

This article focuses on the development of small synthetic inhibitors of cysteine proteases since 1990. The emphasis is on the design of selective cysteine protease inhibitors as research tools and as potential therapeutic agents. The mechanisms of inhibition of the new compounds, where appropriate, are discussed, and also how a knowledge of the differences in mechanisms between cysteine and serine proteases may be exploited in the design of selective inhibitors.

Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone. Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity. An analog of MCoTI-II is one of the most potent inhibitors of matriptase. These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.

The phosphatidylinositol 3‐phosphate 5‐kinase (PIKfyve), is a key enzyme in the phosphoinositide signaling pathway, which plays a crucial role in the development of various cancers and viral diseases. The essential role of small molecules that inhibit this enzyme is limited, which further complicates the design of effective and selective inhibitors. In this study, we employed a BREED‐based de novo ligand design strategy after the detection of a binding pocket in PIKfyve. The BREED‐based molecules were subjected to pharmacokinetic and docking studies. From molecular dynamics (MD) simulations, the hit molecules were observed to exhibit stable behavior in complex with the protein and retain favorable interactions at the binding site. These molecules were also assessed on the synthesizability scale, through reaction‐based enumeration, to determine their ease of synthesis. Overall, the study undertaken describes a de novo hybridization approach that automates the inhibitor design process to reduce the time for producing reliable results when there is limited information about inhibitors in the literature. The selected candidate molecules reveal molecular insights into the inhibition mechanism of PIKfyve.

Exploiting an Asp-Glu “Switch” in Glycogen Synthase Kinase 3 to Design Paralog Selective Inhibitors for Acute Myeloid Leukemia Treatment

Chemotherapy is one of the most effective approaches for cancer treatment. However, to improve efficacy, the therapeutic targets should be identified and characterised. Moreover, new drugs need to be discovered and developed to target different cancer pathways. Current therapeutics can eliminate most of the cancer cells. However, recurrence and metastasis still remain a major failure of cancer therapy. Emerging evidence demonstrates that multidrug resistance (MDR) and the existence of cancer stem cells (CSCs) are two major contributors for the failure of chemotherapy. MDR is a phenomenon in which cancer cells become resistant to structurally and functionally unrelated anticancer agents. CSCs are a small population of cells within cancer cells with capacity for self-renewal, tumor metastasis and differentiation. CSCs are also believed to be associated with chemoresistance. Thus, MDR and CSCs are the greatest challenges for cancer chemotherapy. Significant effort has been made to search for agents that specifically target MDR cells and CSCs. Consequently, some agents derived from nature have been developed to overcome MDR and CSCs. However, the developed chemotherapeutics cannot be used for all the cancers and some of them display severe cytotoxicity. Hence, there is an urgency to investigate the mechanism of drug resistance and to characterise cancer stem cells to identify potential new therapeutic targets. Natural products lie in the heart of the drug discovery. The developed chemotherapeutic compounds mainly originates from the secondary metabolites of microbes, terrestrial plants and marine organisms. In this study, MDR cancer cells were derived from tissue cultured cancer cells by the treatment the cells with fluorouracil (5-FU) and cisplatin (CDDP). CSCs were developed by treatment in serum-free medium with different factors. Fractions and compounds from Nature Bank (Griffith Institute for Drug Discovery, Griffith University), Compounds Australia (Griffith Institute for Drug Discovery, Griffith University) and Traditional Chinese Medicine (TCM) were screening by high through-put screening (HTS). As a result, one potential anticancer flavonoid was isolated from the Australian plant Cryptocarya (QID025519) which was identified by NMR spectroscopic data, in combination with LC-MS. Extracts, fractions and isolated pure compounds from Bruguiera gymnorrhiza andSchisandraviridis were identified as potential agents for the treatment of tongue cancer and breast cancer. The DCM and MeOH extracts and HPLC fractions of B. gymnorrhiza showed antiproliferation activity against cancer cells in a concentration-dependent manner. Further purification of the active fractions led to the isolation of five flavonoids namely rutin, myricetin 3-rutinoside, methoxyflavone, 5-Methoxyluteolin, and 7,3',4',5'-tetrahydroxy-5- gramrione. All five compounds showed antiproliferation activity against CAL27 and MCF7 and MDR cells in a concentration-dependent manner. Methoxyflavone demonstrated the strongest anticancer potential against CAL27 cells, MCF7 cells, CAL27 MDR cells while 7,3',4',5'-tetrahydroxy-5- gramrione illustrated the highest inhibitory effect on MCF7 MDR cells. Both aqueous and ethanol extracts showed activities against MCF7 and CAL27 cancer cells. Bioassay-guided fractionation and purification of the extracts from S.viridis resulted in six active principles, including five dibenzocyclooctene lignans namely gomisin H (1), schisandrin (2), angeloylgomisin H (3), (+)-gomisin M2 (4) and rubschisandrin (5), and one terpenoid, schisanol (6). Compounds 1-3 showed moderate anticancer activities with an IC50 value ranging from 100-200 μg/mL against MCF7 and CAL27 cell lines. Dioxane containing lignans 4-5 and triterpenoid 6 were 10 times more active with IC50 values of14.5, 13.4, 10.6 μg/mL against MCF7, and 21.2, 17.9, 11.7 μg/mL against CAL27, respectively. In addition, two compounds from Compounds Australia exhibited a potential application prospects for tongue cancer and breast cancer therapy. One compound SN00802961 exhibited significant inhibition on MCF7 cells, but low inhibitory effects on fibroblast cells. Meanwhile, it exhibited moderate inhibition on CAL27 MDR cells, CAL27 cells and CSCs. Compound SN00802961 has potently targeted the MAPK/ERK1/2 signaling pathway to induce cytotoxicity in MCF7 cells. Another agent SN00771077 for breast cancer cells in vitro was investigated. The effects of compound SN00771077 on cell viability in vitro were evaluated by treatment of MCF-7 and T47D cells. An in vitro viability assay demonstrated that compound SN00771077 inhibited the cell growth in a dose-dependent manner. The antiproliferative activity of compound SN00771077 is related to its activity on monomeric actin and the subsequent inhibition of polymerization of G-actin monomers. Exposure to compound SN00771077 induced the inhibition of Raf/MEK/ERK pathway in T47D cells. All the results indicated that compound SN00771077 had a strong cytotoxic effects on cancer cells, and shows potential in the treatment of breast cancer by causing the depolymerizing actin cytoskeleton.

The flap region in aspartic proteases is a unique structural feature to this class of enzymes, and found to have a profound impact on protein overall structure, function, and dynamics. Understanding the structure and dynamic behavior of the flap regions is crucial in the design of selective inhibitors against aspartic proteases. Cathepsin-D, an aspartic protease enzyme, has been implicated in a long list of degenerative diseases as well as breast cancer progression. Presented herein, for the first time, is a comprehensive description of the conformational flap dynamics of cathepsin-D using a comparative 50 ns "multiple" molecular dynamics simulations. Diverse collective metrics were proposed to accurately define flap dynamics. These are distance d1 between the flap tips residues (Gly79 and Met301); dihedral angle ϕ; in addition to TriCα angles Gly79-Asp33-Asp223, θ1 , and Gly79-Asp223-Met301, θ2 . The maximum distance attained throughout the simulation was 17.42 and 11.47 Å for apo and bound cathepsin-D, respectively, while the minimum distance observed was 8.75 and 6.32 Å for apo and bound cathepsin-D, respectively. The movement of the flap as well as the twist of the active pocket can properly be explained by measuring the angle, θ1 , between Gly79-Asp33-Met301 and correlating it with the distance Cα of the flap tip residues. The asymmetrical opening of the binding cavity was best described by the large shift of -6.26° to +20.94° in the dihedral angle, ϕ, corresponding to the full opening of the flap at a range of 31-33 ns. A wide-range of post-dynamic analyses was also applied in this report to supplement our findings. We believe that this report would augment current efforts in designing potent structure-based inhibitors against cathepsin-D in the treatment of breast cancer and other degenerative diseases. J. Cell. Biochem. 117: 2643-2657, 2016. © 2016 Wiley Periodicals, Inc.

Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. Caspases, a family of cysteine-dependent aspartate-directed proteases, play a critical role in the initiation and execution of apoptosis. In this study, cytotoxicity and apoptogenic effect of 2-phenyl 4-carboxamide derivatives were evaluated in SKNMC (human neuroblastoma), MCF-7 (human breast adenocarcinoma) and HT-29 (human colon cancer) cell lines. Cell viability was determined by MTT assay. Also, activation of caspase-3 was evaluated by spectrophotometry. The overall cytotoxicity profiles of derivatives demonstrated that the HT-29 cell line has more sensitivity respect to other cell lines. Moreover, our observations indicated that 3-F and 2-F derivatives and 4-Cl derivative increased caspase-3 activation in three carcinoma cell line compared to control. Collectively, these findings suggest that these derivatives are able to induce apoptosis in cancer cell lines.

Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme involved in 1 C metabolism that is upregulated in various cancer cells, but absent in normal proliferating cells. Xanthine derivatives are the first selective inhibitors of MTHFD2 which bind to its allosteric site. Xanthine derivatives (including the co‐crystallized inhibitors) were herein interrogated by molecular/induced‐fit docking, MM‐GBSA binding free energy calculations and molecular dynamics simulations in both MTHFD2 and MTHFD1 (a close homolog expressed in healthy cells). The gained insights from our in silico protocol allowed us to study binding mode, key protein‐ligand interactions and dynamic movement of the allosteric inhibitors, correlating with their experimental binding affinities, biological activities and selectivity for MTHFD2. The reported conformational changes with MTHFD2 upon binding of xanthine derivatives were furthermore evaluated and confirmed by RMSF analyses of the MD simulation trajectories. The results reported herein are expected to benefit in the rational design of selective MTHFD2 allosteric inhibitors.

Introduction: A main choice for cancer treatment is radiotherapy. But, the radiotherapy disadvantage is damages caused by radiation given to normal tissues/organs surrounding cancer. One way to avoid this is via increasing radiosensitization of cancer cells. Gold nanoparticles (GNPs) have shown sensitizing effect on cancer cells by enhancing their absorbed dose. Unlike earlier delivery techniques developed for nanotherapeutics, active targeting can achieve specific effect and higher uptake of GNPs in tumors while leave healthy cells untouched and consequently improve the therapeutic index. To achieve active targeting, GNPs should be equipped with functional ligands which can recognize and adhere to the receptors of cancer cells. Aptamers are small DNA-molecule/RNA-fragments with high specificity and affinity towards target molecules. AS1411 aptamer can specifically bind to over-expressed nucleolin on the membrane of tumor cells including breast cancer. This aptamer is capable to enter cancer cells through specific ligand–receptor interaction. Greater uptake of GNPs by cells may induce increased radiation effects. Cancer stem cells are a small population of cells within a tumor capable for self-renewal and differentiation into various cell types. Materials and Methods: We hypothesized that conjugation of GNPs with AS1411 (AS1411/GNPs) could increase GNPs-mediated radiosensitization in breast cancer cells. We hypothesized that AS1411/GNPs would radiosensitize breast cancer stem/progenitor cells grown to three- dimensional (3D) mammospheres. Cytotoxicity studies of the GNPs and AS1411/GNPs were done on two different cancer cell lines of MCF-7 and MDA-MB-231 with MTT assay. Atomic absorption spectroscopy (AAS) confirmed the cellular uptake of particles. Radiosensitizing effect of GNPs and AS1411/GNPs on MDA-MB 231 and MCF- 7 cells assessed by clonogenic assay. Results: Clonogenic survival data revealed that AS1411/GNPs at 12.5 mg/L results in radiosensitization of breast cancer cells. Mammosphere of MCF-7 was more resistant than their monolayer counterparts. Conclusion: The combination of the sensitizing GNPs with the AS14411 aptamer can be regarded for improved treatment of breast cancer cells especially for the mammosphere MCF-7 cancer cells mimicking cancerous tumors.

Overactive matrix metalloproteinases (MMPs) are associated with a variety of disease states. Therefore, their inhibition is a highly desirable goal. Yet, more than a decade of worldwide activity has not produced even one clinically useful inhibitor. Because of the crucial role of zinc in the activity of the enzyme, the design of inhibitors is usually based upon a so-called zinc binding group (ZBG). Yet, many of the hitherto synthesized potent inhibitors failed clinically, presumably because they bind stronger to metals other than zinc. We have developed in vivo potent inhibitors based on the carbamoylphosphonic group as a putative ZBG. In this paper we report stability constants for Ca(II), Mg(II), Zn(II) and Cu(II) complexes of two potent, in vivo active, MMP inhibitors, cyclopentylcarbamoylphosphonic acid (1) and 2-( N, N-dimethylamino)ethylcarbamoylphosphonic acid (2). Precipitation prevented the determination of stability constants for iron(III) complexes of1 and2. For comparison with carbamoylphosphonates1 and2, we synthesized 2-cyclohexyl-1,1-difluoroethylphosphonic acid (3), which does not inhibit MMP, and determined the stability constants of its complexes with Mg(II), Ca(II) and Zn(II). Comparison with the values obtained from the complexes of1 and2 with those from3 indicates participation of the C=O group in the metal binding of the former compounds. The complex stability orders for both1 and2 are Ca(II)<Mg(II)<Zn(II)<Cu(II). In addition, the results indicate that at pH>8 the dimethylamino group of compound2 can also participate in the binding of the transition metals Cu and Zn. On the other hand, the amino group in carbamoylphosphonic acid2 lowers the stability of the complexes with metals favoring oxygen ligands (Ca, Mg and Fe) and increases the selectivity towards Zn. These results are helpful for rationalizing the results observed on our MMP inhibitors hitherto examined, and are expected to be useful for the design of new selective inhibitors.

According to the statistic data of cancer in China, breast cancer has the highest incidence in female residents in cities of China. Although the traditional therapies, including surgery, radiation therapy, chemotherapy and hormone therapy have led to the increased 5-year and 10-year survival, there are still a small number of cancer cells survived from treatment, causing cancer recurrence and/or metastasis. Around 70% of survived breast cancer can metastasize to the lung, 60% to the bone and liver and 15% to the brain. Breast cancer stem cells, a subpopulation of breast cancer cells carrying stem cell properties, have strong ability to regenerate tumor and natural resistance to radiation and chemotherapy, which may be responsible for cancer recurrence and metastasis. The strategies targeting at cancer stem cell bring new hope for the treatment of cancer in the near future. In human breast cancer tissues, ALDH positive cells and CD44+ CD24- Lin- cells have been identified as breast cancer stem cells. In this review, we discussed the identification, origin, properties, regulatory mechanism of breast cancer stem cells and the resistance against radiation and chemotherapy, as well as the advances of clinical targeted therapy. Key words: Breast neoplasms; Neoplastic stem cells; Radiotherapy; Chemotherapy; Drug resistance

Arming Antibodies for Cancer Therapy: Transglutaminase-Mediated Toxin Conjugation

Cancer is still a leading cause of death worldwide. Despite the progress in the molecular understanding of cancer diseases, there’s an urgent need in novel therapeutics and drug delivery strategies. Many novel anti-cancer compounds in early development are characterized by unfavorable physico-chemical properties and lack in drug-like properties. As a result, many of these compounds suffer from insufficient pharmacokinetic properties and show a high accumulation in off- target tissue that can induce dose-limiting side effects. Nanomedicines depict a promising strategy to optimize the pharmacokinetics of such compounds and to deliver them to their site of action: The cancer cell. The goal of this thesis was to develop nanoparticulate drug delivery platforms for passive and active drug targeting. In addition, a novel nanoparticle-based gene therapeutic for the treatment of liver cancer was evaluated. This thesis can be summarized in two main parts as follows: In a first part, a biocompatible and biodegradable polymer was used to prepare micelles for the delivery of small molecular anticancer drugs. These micelles were tested subsequently on in vitro and in vivo models. A highly reproducible protocol for the formulation of doxorubicin-loaded micelles was developed and micelles were characterized extensively for their physico-chemical properties. Cellular uptake of micelles was analyzed and their therapeutic potential was assessed in vitro on human cancer cells. To passively accumulate in solid tumors, nanoparticles need to be long-circulating and must remain in the blood circulation for hours. Therefore, the pharmacokinetic profile and biodistribution of doxorubicin-loaded micelles in rats was analyzed and compared to the gold standard of long-circulating nanoparticles: PEGylated liposomes. In a next step, a protocol for the preparation of so-called gold-nanohybrids was developed. Such nanohybrids are valuable tools to analyze nanoparticle-cell interactions and the intracellular fate of nanoparticles in detail. Further, such nanoparticles can be used as diagnostic tools. In a last step, micelles were functionalized with an antibody for targeted drug delivery. Cellular internalization of these micelles was analyzed using an array of methods. In a second part, a novel therapeutic strategy using the main effector protein of the rat parvovirus (H-1) for the treatment of hepatocellular carcinoma (HCC) was developed. H-1 parvovirus showed promising results in the preclinical setting and was consequently tested in a clinical trial in patients suffering form glioma. Despite this development, viral therapies may be linked with several issues. Therefore, the potential of the viral effector protein NS1 for the treatment of liver cancer was analyzed after non-viral gene delivery. In a first step, the gene-delivery efficiency and the therapeutic effect were analyzed in a panel of human liver cancer-derived cell lines. Various in vitro assays were used to study the NS1-induced cell death in detail. To show that this therapeutic approach is specific for cancer cells, the treatment was furthermore tested on healthy human liver cells. To identify cells that are susceptible for this therapeutic approach, a biomarker for the sensitivity to non-viral NS1 therapy was evaluated. Finally, safety of this therapy was analyzed in mice after single and multiple dosing.

Inhibitors of aldehyde dehydrogenases of the 1A subfamily as putative anticancer agents: Kinetic characterization and effect on human cancer cells